Enhancement of developmental competence after in vitro fertilization of porcine oocytes by treatment with ascorbic acid 2-O-alpha-glucoside during in vitro maturation.

The present study was conducted to examine the effect of ascorbic acid 2-O-alpha-glucoside (AA-2G), a stable ascorbate derivative, on the sustenance of cytoplasmic maturation responsible for subsequent developmental competence after in vitro fertilization of porcine oocytes. Cumulus-oocyte complexes were cultured for 44 h in North Carolina State University 37 medium supplemented with cysteine, gonadotropins, 10% (v:v) porcine follicular fluid, and 0-750 microM AA-2G. When oocytes were matured in the presence of 250 microM AA-2G, their ability to promote transformation of the sperm nucleus into the male pronucleus (MPN) was strongly enhanced after in vitro fertilization. Similarly, the presence of 25 microM beta-mercaptoethanol (ME) enhanced the degree of progression to MPN of penetrated sperm by associating with the increase in intracellular glutathione (GSH) content. Although the AA-2G treatment during oocyte maturation showed no influence on the GSH concentration, significantly higher levels of ascorbic acid (AsA) were detected in these oocytes than in those oocytes cultured without AA-2G (P < 0.05). The length of DNA migration encompassed by reactive oxygen species (ROS), generated by the hypoxanthine-xanthine oxidase system, was not increased in the oocytes treated with AA-2G, whereas ME treatment could not block the DNA damage by ROS. These findings indicate that AA-2G in maturation medium can potentiate the cellular protection of oocytes against oxidative stress by continuously supplying AsA. The proportion of development to the blastocyst stage after in vitro insemination was significantly increased in oocytes matured with AA-2G (P < 0.05), and this proportion showed no difference in comparison with that of oocytes treated with ME. These findings suggest that a critical concentration of intracellular AsA, supplied by AA-2G during in vitro maturation, plays an important role in supporting the cytoplasmic maturation responsible for developmental competence after fertilization by prevention of oxidative stress against porcine oocytes.     

Insulin release from BK virus-induced and in vitro maintained insulinoma cells of Syrian golden hamster

The pancreatic tumor cells (In 111) derived from BK virus-induced insulinoma of Syrian golden hamsters were maintained in culture for several passages and were studied for their insulin secretory ability under various stimulatory conditions. Insulin release was not increased by D-glucose stimulation (27.8 mM), while dibutyryl cyclic AMP (1 mM), theophylline (1 mM), 3-isobutyl-l-methylxanthine (0.1 mM) and elevation of medium calcium from 0.5 to 2.7 mM stimulated insulin release 2.5- to 4-fold. There was a concomitant increase of medium cyclic AMP with addition of theophylline. Streptozotocin (2 mM) treatment for 48 hours significantly reduced insulin release, while alloxan (2 mM), had no inhibitory effect on insulin release. The results indicate that while in vitro-maintained islet tumor cells, In 111, have a cyclic AMP-mediated process involved in insulin secretion analogous to normal beta cells, these cells lack the ability to recognize glucose as an insulin secretagogue probably due to a defect in the cell membrane, though the possibility of alteration in glucose metabolism cannot be fully excluded.     

In vitro effects of glucocorticoid on glucose transport in rat adipocytes: evidence of a post-receptor coupling defect in insulin action

The in vitro effect of glucocorticoid on insulin binding and glucose transport was studied with rat adipocytes. Isolated rat adipocytes were incubated with or without 0.70 microgram/ml (1.9 mumol) of hydrocortisone in TCM 199 medium at 37 degrees C, 5% CO2/95% air (v/v), pH 7.4, for 2, 4, and 8 h, and then fat cell insulin binding and insulin-stimulated 3-O-methylglucose transport were measured. Hydrocortisone did not affect insulin binding in terms of affinity or receptor number. Glucose transport in the absence of insulin was significantly decreased at the incubation time of 2 h and continued to decrease up to 8 h of incubation with hydrocortisone. Decreased insulin sensitivity of glucose transport (i.e., a right-ward shift of the dose response curve) was also demonstrated after 2 h incubation with hydrocortisone, and the ED50 of insulin was maximally increased at 4 h of incubation (0.53 ng/ml for treated vs. 0.22 ng/ml for control cells). Maximal insulin responsiveness was also significantly decreased in treated cells after 8 h incubation with hydrocortisone. When percent maximum glucose transport was expressed relative to receptor-bound insulin, the ED50 values of treated and control cells were 10.5 and 7.2 pg of bound insulin, per 2 X 10(5) cells, respectively. Thus, it was evident that glucocorticoid induced a post-receptor coupling defect in the signal transmission of insulin-receptor complex.     

N-acetylgalactosaminyl disaccharide terminals contained in presumed carbohydrate epitopes specific to sea-squirt allergy

Using the methods of methylation/GLC, MS, and 1H-NMR analyses, a disaccharide structure of GalNAc alpha 1----2Fuc1---- was identified as one of the major structural units at non-reducing terminals of a highly branched and N-glycosidically linked carbohydrate on an allergenically active glycopeptide, Gp-2 (Mr ca. 8,000). Gp-2 was one of the major fractions in the Pronase digest of a sea-squirt antigen, Gi-rep, and is capable of eliciting the skin reaction specific to patients with sea-squirt allergy similarly to Gi-rep. An additional disaccharide structure of GalNAc1----(3/4)HexNAc1---- was also expected as the other major non-reducing terminal unit, though the anomeric configuration of the terminal GalNAc could not be identified. As Gp-2 carbohydrate was found to contain 3 mol each of the two types of terminal GalNAc-containing units, both were nominated as possible components of the IO4- oxidation-sensitive epitopes which are responsible for the allergenic activity in Gp-2 and its mother antigen, Gi-rep. A few moles of Fuc and Gal were also detected as minor non-reducing terminals in addition to the 6 mol of the GalNAc-containing units.     

Structure of an allergenic pentasaccharitol, Gp-1 beta-b6, isolated from a sea squirt antigen, Gi-rep, as a minimum structural unit responsible for its allergenicity

An allergenic pentasaccharitol, Gp-1 beta-b6, was isolated as a minimum structural unit responsible for the allergic reaction in skin of patients with sea squirt allergy from a saccharitol fraction, Gp-1 beta-b, that had been liberated by beta-elimination from a glycopeptide in a Pronase digest of a sea squirt antigen, Gi-rep. Methylation/GC-MS and FAB-MS analyses indicated the sugar sequence of Gp-1 beta-b6 to be GalNAcl----2Fucl----(GalNAc1----) 3,4GlcNAc1----3GalNAc-ol. To analyze the structure in more detail, Gp-1 beta-b6 was labeled with p-aminobenzoic acid ethyl ester (ABEE), i.e., the reducing terminal 3-O-substituted GalNAc-ol of the saccharitol was oxidized to 2-O-substituted L-ThrNAc with equimolar periodate, and the resultant aldehyde was labeled with ABEE by reductive amination. The ABEE-labeled Gp-1 beta-b6 was subjected to sequential exoglycosidase digestion with beta-N-acetylhexosaminidase, alpha-N-acetylgalactosaminidase, and alpha-fucosidase, and the digests were chromatographed on an HPLC column of TSK gel Amide 80. From the results of the HPLC, methylation/GC-MS, and FAB-MS analyses of the digests of the labeled substrate, the structure of Gp-1 beta-b6 was determined to be GalNAc alpha 1----2Fuc alpha 1----3(GalNAc beta 1----4)GlcNAc beta 1----3GalNAc-ol. Enzymatic elimination of either the non-reducing terminal beta-GalNAc or the non-reducing terminal alpha-GalNAc led to inactivation of the allergenic pentasaccharitol. Accordingly, it is possible that the allergenic saccharitol contains two disaccharide units as the allergy-specific epitopes, one GalNAc alpha 1----2Fuc alpha 1---- and the other GlcNAc beta 1----4GLcNAc beta 1----.     

Crown profile of foliage area characterized with the Weibull distribution in a hinoki (Chamaecyparis obtusa) stand

Summary Thirten sample trees of various sizes in a 29-year-old hinoki [Chamaecyparis obtusa (Sieb, et Zucc.) Endl.] plantation were felled and subjected to the stratified clip technique. Crown profile of foliage area fitted well with the Weibull distribution. The crown profile tended to be more skewed toward the top of crowns in smaller trees than in larger trees. This tendency was reflected in the value of the shape parameter of the Weibull distribution. The shape parameter ranged from 1.73 to 3.23 and gradually increased up to an asymptotic value with an increase of stem diameter at breast height. The scale parameter of the distribution ranged from 1.0 to 4.2 and tended to increase in proportion to stem diameter at breast height. Foliage area of a tree correlated well with stem diameter at breast height through an ordinary allometric equation. Tree height could be approximated fairly well by a generalized allometric equation as a function of stem diameter at breast height. On the basis of the census of stem diameter at breast height, canopy profile could be constructed synthesizing crown profiles of foliage area for individual trees in the stand. Leaf area index was estimated to be 6.6 ha ha^–1.     

The effect of high-glucose condition on insulin binding to IM-9 lymphocytes

The long-term effect of high-glucose condition on insulin binding to IM-9 cells was studied by incubating the cells in RPMI-1640 medium containing 450 mg/dl of glucose. Insulin binding began to decrease after incubation for 6 days in high-glucose-treated cells, and significantly decreased after 14 days due to the reduction of receptor affinity. The number of binding sites for insulin did not change by the treatment. Thus, high-glucose condition per se does not appear to induce the decrease of the number of insulin receptors on target cells, as observed in diabetic patients.     

Hepatic and intestinal gamma-glutamyltranspeptidase activity: its activation by chronic ethanol administration.

To study the effect of chronic ethanol administration on the activity of gamma-glutamyltranspeptidase (GGTP) in various tissues, female rats were pair-fed liquid diets with 36% of total calories either as ethanol or isocaloric carbohydrate (controls). Six weeks of ethanol feeding in an increase of cytochrome P450 content by 70%. Hepatic microsomal GGTP activity was more than doubled after ethanol feeding whether expressed per gram of liver or per mg of microsomal protein. Furthermore intestinal GGTP activity was significantly enhanced after ethanol, whereas there was no change in the enzyme activity in either kidney or pancreas. Phenobarbital administration to rats also resulted in an enahancement of GGTP activity in the liver but not in the intestine. These results suggest that enhanced hepatic and intestinal GGTP activities may contribute, at least partly, to increased serum GGTP activity frequently seen in alcoholics.